Antiviral potential and stability analysis of chicken interferon-α produced by Newcastle disease virus in chicken embryo fibroblast cells

Chicken interferon-alpha (chIFN-alpha) is an important antiviral cytokine and represents one of the first lines of the chicken's innate immune system. The current study is the first-ever report of chicken IFN (chIFN) production in Pakistan. In this study, we have used live and UV-irradiated Newcastle disease virus (NDV) to induce the expression of chIFN-alpha in chicken embryo fibroblast (CEF) cells. ChIFN-alpha was partially purified in a two-step protocol; ultracentrifugation followed by treatment with anti-chIFN-beta antibodies. The purified chIFN-alpha was analysed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the in vitro antiviral potential of chIFN-alpha was determined against the H9N2 avian influenza virus (AIV) via a cytopathic inhibition assay. The relative mRNA level of the IFN-stimulated genes (ISGs) in the IFN-stimulated CEF cells was measured at various time intervals by a quantitative polymerase chain reaction (qPCR). The stability of natural chIFN-alpha to the temperature, pH, and ultraviolet (UV) light was also determined. The in vivo therapeutic potential of chIFN-alpha was determined in 7-day-old broiler chickens challenged with AIV. We found that a higher chIFN-alpha expression level was induced by the UV-irradiated NDV in the CEF cells as compared to the live NDV. The UV-irradiated NDV induced the maximum IFN production in the CEF cells at 24 h post-infection. Two bands of 21 kDa on SDS-PAGE confirmed the presence of the chIFN-alpha protein. The cytopathic inhibition assay indicated the strong antiviral activity of chIFN-alpha against AIV. Our results of the stability analysis showed that chIFN-alpha was stable at a wide range of temperatures and pH levels. However, a little exposure to UV-light resulted in a significant loss of antiviral activity. We also observed that the antiviral activity of chIFN-alpha is related to the expression levels of the antiviral ISGs. The results of the in vivo study showed that the chIFN-alpha therapy via the oral route resulted in a significant improvement in the tracheal pathology of chickens challenged with AIV. In conclusion, we suggest that chIFN-alpha could be an important therapeutic tool to control avian influenza infection in poultry.